Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species, R. conorii, R. siberica, R. japonica, R. akari, and others cause diseases of lesser severity. Still other species in the spotted fever group, R. montana, R. peacockii, R. belli, and R. rhipicephali, are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. The typhus group of rickettsia, typified by R. prowazeki, the agent of epidemic typhus, include some of the historically most devastating disease agents known to mankind. The typhus group also includes species of lesser or no virulence potential to humans. R. prowazeki and R. rickettsii are classified as Biodefense Catagory B and C agents, respectively. Rickettsia rickettsii is a member of the spotted fever group rickettsiae and the etiologic agent of Rocky Mountain spotted fever (RMSF). R. rickettsii is a small obligate intracellular Gram-negative organism maintained in its tick host through transovarial transmission. Infection with R. rickettsii occurs through the bite of an infected tick. Once the organism gains access to the host it is able to replicate within the host vascular endothelial cells and spread from cell to cell by polymerizing host cell actin. Damage to vascular endothelial cells by R. rickettsii leads to increased vascular permeability and leakage of fluid into the interstices causing the characteristic rash observed in RMSF. Infection with R. rickettsii results in a severe and potentially life threatening disease if not diagnosed and treated properly. While much is known about the progression of disease, the molecular mechanisms involved in the pathogenesis of RMSF are poorly understood. Strains of Rickettsia rickettsii vary dramatically in their virulence in animal model systems and severity of human disease. The obligate intracellular lifestyle of rickettsiae and the lack of tractable genetic systems make it difficult to identify genes involved in virulence. With the completed sequences of multiple rickettsial species, it has become possible to investigate differences between virulent and avirulent strains of rickettsiae through comparative genomics. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, less virulent Morgan and Sao Paulo, moderate virulence for the R strain, low for HLP, and Iowa which causes no fever. Plaque morphologies also vary in size and degree of host cell lysis. To identify genes involved in the virulence of R. rickettsii, the genomes of four different strains were resequenced by comparison to known genome sequences (CGS). R. rickettsii Morgan, HLP, R and Sao Paulo strains were compared to avirulent strain R. rickettsii Iowa and virulent R. rickettsii Sheila Smith. SNP analysis revealed the Montana strains Sheila Smith and R to be highly similar while the Eastern strains Iowa and Morgan were more similar to each other. Sao Paulo appears to be most like the Montana strains, whereas HLP contained too many differences to categorize. The region of ompA containing 13 tandem repeats was sequenced using the transposon system EZ-TN5 revealing only 7 shared SNPs (4 nonsynonymous) for R and Morgan strains compared to Sheila Smith with an additional 16 SNPs identified in Morgan. Analysis of rompB, another major surface antigen, shown in Iowa to have a defect in processing, reveals identical sequences between Iowa and Morgan and R being identical to Sheila Smith. By immunoblotting, both rOmpA and rOmpB appear similar among the more virulent strains, whereas Iowa and HLP do not. The number of coding sequence SNPs between sequences is low, narrowing the field of possible virulence factors. One of the major distinctions is in a major surface antigen and autotransporter, rOmpA. rOmpA is severely truncated in the avirulent strain, Iowa, but present is each of the virulent strains. To determine the role of rOmpA in virulence, a commercial targeted mutagenesis system known as TargeTron was applied to successfully knock out rOmpA. Surprisingly, the knockout strain showed no diminishment of virulence in a Guinea pig model of infection nor any defects in replication or adherence to host cells.